Myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are a group of chronic hematologic malignancies characterized by the overproduction of myeloid lineage cells. The JAK2V617F mutation has been found in ~95% cases of PV and 50-60% cases of ET and PMF However, the contribution of JAK2V617F in these three different MPNs still remains unclear. Using an inducible JAK2V617F knock-in mouse, we have shown that expression of heterozygous JAK2V617F in hematopoietic compartments is sufficient to cause a PV-like disease whereas homozygous JAK2V617F expression accelerates the progression to myelofibrosis (MF). We also have shown that only JAK2V617F-expressing hematopoietic stem cells (HSCs) have the capacity to self-renew and propagate the MPN disease. However, the actual role of JAK2V617F in HSCs has remained unclear and controversial. Most studies are focused on the bone marrow (BM) HSC function. The contribution of spleen HSCs in the maintenance of JAK2V617F-evoked MPN remains unknown. We have found that the number of HSCs in the BM is reduced over time while spleen HSC pool is consistently increased in JAK2V617F knock-in mice. We hypothesize that JAK2V617F expression may cause aberrant HSC function, and JAK2V617F-expressing spleen HSCs may play an important role in the long-term maintenance of MPNs. JAK2 inhibitor therapy exhibits some benefits to the MPN patients but results in significant hematopoietic toxicities since JAK2 is critical for normal hematopoietic development. Identification of the targets of JAK2V617F in HSCs that are required for MPN but are not essential for normal hematopoietic development would facilitate the development of new targeted and safer therapies for MPNs. In preliminary studies, using microarray gene expression profiling, we have identified several targets of JAK2V617F in HSCs. Therefore, Aim 1 will determine the effects of JAK2V617F on BM and spleen HSCs, and identify the downstream targets of JAK2V617F in HSCs that are required for MPN. Although JAK2V617F is the most common mutation found in MF, the mechanisms of MF induced by JAK2V617F remains unknown. We have found that Shp2 is constitutively phosphorylated in JAK2V617F- positive MPN/leukemia patient derived cell lines and in the BM of JAK2V617F knock-in mice. In preliminary studies, we have found that deletion of Shp2 inhibits PV and prevents the development of MF in JAK2V617F mice. We hypothesize that Shp2 may play an important role in the pathogenesis of PV and MF induced by JAK2V617F. Therefore, Aim 2 will define the role of Shp2 in PV and MF induced by JAK2V617F. Inactivating EZH2 mutations have been found in patients with MF. Moreover, a significant proportion of EZH2 mutated MF patients harbor the JAK2V617F mutation. We hypothesize that EZH2 deficiency may contribute to the phenotypic diversity in JAK2V617F-positive MPNs. In Aim 3, we will determine the contribution of EZH2 deficiency in JAK2V617F-evoked MPN. Collectively, these studies will provide important new insights into the molecular pathogenesis of MPNs and may identify new therapeutic targets for treatment of MPNs.